Hope and Challenges: Immunotherapy in EGFR-Mutant NSCLC Patients

EGFR tyrosine kinase inhibitors (TKIs) are the preferred initial treatment for non-small cell lung cancer (NSCLC) patients harboring sensitive EGFR mutations. Sadly, remission is transient, and no approved effective treatment options are available for EGFR-TKI-advanced EGFR-mutant NSCLCs. Although immunotherapy with immune checkpoint inhibitors (ICIs) induces sustained cancer remission in a subset of NSCLCs, ICI therapy exhibits limited activity in most EGFR-mutant NSCLCs. Mechanistically, the strong oncogenic EGFR signaling in EGFR-mutant NSCLCs contributes to a non-inflamed tumor immune microenvironment (TIME), characterized by a limited number of CD8+ T cell infiltration, a high number of regulatory CD4+ T cells, and an increased number of inactivated infiltrated T cells. Additionally, EGFR-mutant NSCLC patients are generally non-smokers with low levels of PD-L1 expression and tumor mutation burden. Promisingly, a small population of EGFR-mutant NSCLCs still durably respond to ICI therapy. The hope of ICI therapy from pre-clinical studies and clinical trials is reviewed in EGFR-mutant NSCLCs. The challenges of application ICI therapy in EGFR-mutant NSCLCs are also reviewed.


Subtypes of EGFR Alterations May Be Sensitive to ICI Therapy
ICI efficacy varies in the subtypes of EGFR alterations in EGFR-mutant NSCLCs.EGFR wild-type and L858R NSCLCs had a similar outcome, while EGFR 19del NSCLCs had a worse outcome when treated with ICI therapy [43].Other groups also supported the findings that ICI therapy alone or in combination exhibited improved survival outcomes in the EGFR L858R group relative to the EGFR 19del group [44][45][46][47].One of the possible explanations was that EGFR L858R-mutant tumors were more likely to have a significantly increased CD8 + T cell infiltration than EGFR 19del tumors [39].Additionally, EGFR T790Mnegative patients were more likely to benefit from ICI therapy than EGFR T790M-positive patients [48,49], as the EGFR T790M-negative tumors had higher PD-L1 expression than the EGFR T790M-positive tumors [48,50].Other than the common mutations, 10-20% of EGFR-mutant NSCLCs harbor non-EGFR L858R or 19del mutations, known as uncommon mutations, such as S768I and G719X [5,37,51,52].Relative to common EGFR mutations, uncommon EGFR-mutant tumors were likely to express high PD-L1 (≥50%), high TMB, and a high infiltration of CD8 + T cells [37,49,52,53].Of the uncommon EGFR-mutant NSCLCs with dual high PD-L1 expression and abundant CD8 + T cell infiltration, 36.7% showed a favorable response to ICI therapy [49,53].

Heavily Pre-Treated EGFR-Mutant NSCLCs Are Likely to Respond to ICI Therapy Relative to Treatment-Naïve EGFR-Mutant NSCLCs
EGFR-TKI treatment results in dynamic changes in host immunity.EGFR-TKI-advanced EGFR-mutant NSCLC patients trended to have concurrent high levels of PD-L1 expression and a high number of CD8 + T cell infiltration [34].The proportion of EGFR-mutant NSCLCs with high PD-L1 (≥50%) increased from 14% to 28% after EGFR-TKI treatment [54].Heavily pre-treated EGFR-TKI-advanced EGFR-mutant NSCLCs with high expression of PD-L1 (≥25% of tumor cells) had a better median OS (mOS) than that with low expression of PD-L1 (<25% of tumor cells) (13.3 vs. 9.9 months) after ICI therapy in a Phase II ATLANTIC study [29,55].Impressively, two of five EGFR-TKI-advanced EGFR-mutant NSCLCs with increased PD-L1 after an EGFR blockade showed a durable response to subsequent ICI therapy [54].Overall, EGFR-TKI-advanced EGFR-mutant NSCLCs may be the groups responding to ICI therapy.However, ICI therapy immediately followed by EGFR TKI therapy may cause immune-related adverse effects (irAEs), as discussed below.

IrAEs Observed during ICI Therapy Are Associated with Efficacy
A growing body of literature supports the theory that the occurrence of irAEs during ICI therapy is associated with improved treatment efficacy.A retrospective analysis proved that the 270 metastatic NSCLC patients who experienced irAEs after ICI therapy had improved PFS, overall response rate (ORR), disease control rate (DCR), and OS relative to those who did not experience irAEs (PFS: 5.2 vs. 1.97 months; ORR: 22.9% vs. 5.7%; DCR: 76% vs. 58%; OS: not reached vs. 8.21 months) [56].Similarly, secondary analysis of the Phase I CA209-003 clinical trial conducted at 13 US medical centers led to the finding that the mOS was significantly longer among patients with irAEs of any grade (19.8 months; 95% CI, 13.8-26.9months) or grade 3 or more (20.3months; 95% CI, 12.5-44.9months) compared with those without treatment-related AEs (5.8 months; 95% CI, 4.6-7.8months) (p < 0.001 for both comparisons based on hazard ratios) [21].Another retrospective study found that around 43.6% of 195 advanced NSCLCs treated with ICI therapy experienced irAEs, and those NSCLCs with irAEs had significantly longer mPFS (5.7 s. 2.0 months), PFS (8.5 vs. 4.6 months), mOS (17.8 vs. 4.0 months), and OS (26.8 vs. 11.9 months) than those who did not experience irAEs [57].In a prospective study with 43 advanced NSCLCs, earlier irAEs were associated with better objective response and disease control rates than those without irAEs after ICI therapy (37% vs. 17% and 74% vs. 29%, respectively) [58].Another prospective study with 76 advanced NSCLCs also found that NSCLCs who experienced irAEs within two weeks of beginning ICI therapy had significantly longer mPFS than those who did not (5.0 vs. 2.0 months) [59].However, it is challenging to predict irAEs, and severe irAEs may be life-threatening.A further understanding of irAEs during the application of immunotherapy is necessary.
PD-L1 was a predictive and prognostic biomarker for response to immunotherapy in NSCLCs [25].Several clinical trials reported the association between PD-L1 expression and clinical outcomes in NSCLC patients [25,33,89].However, the predictive and prognostic roles for PD-L1 in immunotherapy were limited in EGFR-mutant NSCLCs [39].ICI therapy did not show clinical benefits in TKI-naïve EGFR-mutant NSCLCs, even with high PD-L1 expression.A Phase II trial of ICI therapy in treatment-naïve EGFR-mutant NSCLCs was discontinued due to lack of efficacy despite 73% of enrolled patients with more than 50% PD-L1 expression [33,89].Patients with low or even undetectable PD-L1 expression also had improved survival with ICI therapy vs. chemotherapy [25].Cross-comparison is sometimes challenging due to various immunohistochemical (IHC) assays with different scoring systems and cutoff values [90,91], making a universal assay for assessing PD-L1 expression with appropriate cutoff points important.
PD-L1 expression levels might not directly reflect the underlying T cell activity in EGFR-mutant NSCLCs [39].PD-L1 was expressed in tumor and circulating immune cells, such as dendritic cells and myeloid-derived immune suppressor cells [92,93].In POPLAR and OAK trials, assessing PD-L1 expression in tumor cells and tumor-infiltrating immune cells led to the finding that higher PD-L1 levels in both tumor cells and tumor-infiltrating immune cells were associated with improved patient survival after ICI therapy [25,94].Patients with more than 30% PD-L1 + CD11b + myeloid cells before ICI therapy showed a 50% superior response rate [95].Not only PD-L1 could bind with PD-1 to negatively regulate T cell activity, PD-L2 was another ligand for PD-1 with 2-6-fold higher affinity to PD-1 than PD-L1 [96,97].EGFR signaling also regulated PD-L2 expression in NSCLCs [98].

EGFR-Mutant NSCLCs Are Non-Smokers, Generally
Tobacco is a known risk factor for lung cancer.However, PD-L1 positivity was also associated with smoking history [79][80][81], and smokers were more likely to benefit from ICI therapy [103][104][105].Although EGFR mutations are more enriched in never-smokers, EGFR mutations in NSCLCs were also found in ever-smokers [6,[106][107][108].Compared with common EGFR mutations, uncommon EGFR mutations were significantly associated with smoking [109].These findings further support the above-discussed findings that the NSCLCs with uncommon EGFR mutations were more likely to respond favorably to ICI therapy than NSCLCs with common EGFR mutations [49,53].

EGFR-Mutant NSCLCs Have a Lymphocyte-Depleted TIME
Crosstalk between cancer cells and TIME is a hot research topic with the rapid development of immunotherapy in cancers, including lung cancer.Based on the presence or absence of tumor-infiltrating lymphocytes (TILs), there were four different types of TIME in tumors: TIL + PD-L1 + , TIL -PD-L1 − , TIL -PD-L1 + , and TIL + PD-L1 − .However, only TIL + PD-L1 + tumors with lymphocyte infiltration and PD-L1 expression responded to ICI therapy [110].To better reflect the complex relationship of the tumor, host, and environmental factors, tumors were also classified into the following types: the immune-desert tumor, the immune-excluded tumor, and the inflamed tumor [111].The immune-desert and immune-excluded tumors were naturally resistant to ICI therapy.EGFR-mutant tumors had a "lymphocyte depletion" phenotype [34,36,38,112,113], characterized by a pronounced lack of the infiltration of CD8 + T cells [34,36,38,114], suggesting an immunosuppressive TIME.Single-cell RNA sequencing further confirmed the low T cell infiltration in the TIME of treatment-naïve EGFR-mutant NSCLCs [115].Additionally, EGFR-mutant NSCLC tumors had markedly less crosstalk between T cells and other cell types via the PD-1/PD-L1 pathway than EGFR-negative NSCLCs [116].
Tumors without detectable EGFR expression responded to EGFR inhibition [128], implicating that EGFR blockade potentially influenced the tumor-specific immune responses.Deficient Egfr in murine myeloid cells decreased carcinogenesis, suggesting a tumor-promoting function by myeloid cell-intrinsic EGFR signaling [129].Macrophages in the TIME expressed EGFR [129,130].EGF secreted by tumor cells promoted an M2 polarization of tumor-associated macrophages (TAMs), associated with the suppression of cytotoxic T cell function [131].In contrast to M2-type TAMs, higher infiltrated M1-type TAMs found in NSCLC with uncommon EGFR mutations (G719X and exon 20s) correlated with longer PFS than common EGFR mutations [37].
EGFR-mutant NSCLC cell lines significantly downregulated MHC class I molecule expression compared with the EGFR wild-type NSCLC cell lines in response to IFNγ [132].PI3K-AKT and MAPK pathways, the primary downstream signaling pathways of EGFR [133,134], also suppressed MHC class I molecule expression [132,[135][136][137][138]. The data suggested that the downregulated MHC class I molecules were through abnormal EGFR signaling in EGFR-mutant NSCLCs.Indeed, EGFR inhibition augmented the expression of MHC class I molecules [139-141].

EGFR-Mutant NSCLCs Respond Poorly to ICI Therapy Alone or in Combination
EGFR-mutant NSCLCs generally responded poorly to ICI therapy [30,34].In the Phase III CheckMate-057 clinical trial, subgroup analysis of the patients with activating EGFR mutations revealed no PFS and OS benefit from ICI therapy [23].Subgroup analysis of Phase III KEYNOTE-010 clinical trial data showed no improved OS benefit from ICI therapy in EGFR-mutant NSCLCs [27].Similarly, EGFR-mutant NSCLCs did not achieve prolonged OS from ICI therapy vs. chemotherapy in Phase III OAK clinical trial [25].A retrospective analysis showed that EGFR mutations were associated with low clinical response to ICI blockade in NSCLCs [34].It was confirmed in a pooled analysis from three clinical trials (CheckMate-057, KEYNOTE-010, and POPLAR) that the EGFR wildtype but not the EGFR-mutant NSCLCs had prolonged OS [31].Combining data from five trials (CheckMate-017, CheckMate-057, KEYNOTE-010, OAK, and POPLAR), Lee et al. additionally confirmed no prolonged OS in EGFR-mutant NSCLCs receiving ICI therapy relative to chemotherapy [32].A Phase II trial was halted due to lack of efficacy and two deaths within six months of enrollment to ICI therapy in treatment-naïve EGFRmutant patients [33].One of the two deaths was from pneumonitis [33].In the Phase II WJOG8515L clinical trial, nivolumab was inferior to the chemotherapy with worse mPFS (1.7 vs. 5.6 months) and ORR (9.6% vs. 36%) in EGFR-TKI-advanced EGFR-mutant NSCLCs without a T790M mutation [142].In a retrospective study with 58 EGFR-mutant NSCLCs, patients who responded to prior EGFR TKIs for more than ten months displayed significantly shorter PFS to ICI therapy compared with those who responded to prior EGFR TKIs for less than ten months (1.6 vs. 1.9 months, p = 0.009) [143].Adding ICI therapy to chemotherapy was associated with worse survival than platinum doublet chemotherapy alone in osimertinib-advanced EGFR-mutant NSCLCs [144].Phase III IMpower130 clinical trial also found no benefit in the EGFR-mutant subgroup treated with ICI therapy and chemotherapy combined vs. chemotherapy alone [145].Based on all these negative findings, the National Comprehensive Cancer Network (NCCN) clinical practice guidelines of NSCLC (version 4, 2021) did not recommend immunotherapy for treating EGFR-mutant NSCLCs.

Safety Concerns and Lower Clinical Outcomes Regarding EGFR TKI and ICI Combined for the Treatment of EGFR-Mutant NSCLCs
Compared with chemotherapy, ICI therapy generally correlates with fewer adverse reactions.However, ICI therapy was associated with immune-related adverse events (irAEs), probably due to the disruption of immunologic homeostasis [146].There is a growing concern that a combination of EGFR TKIs, especially osimertinib or gefitinib, and ICI therapy may be associated with an increased risk of toxicity.In the Phase 1/2 KEYNOTE-021 clinical trial, five of seven untreated stage IIIB/IV EGFR-mutant NSCLC patients (71.4%) treated with ICI therapy plus gefitinib had grade 3/4 liver toxicity, leading to permanent treatment discontinuation in four patients [147].A lung adenocarcinoma patient bearing EGFR 19del treated with osimertinib following ICI therapy had Stevens-Johnson syndrome and hepatotoxicity [148].In a Phase Ib TATTON clinical trial, 38.5-60% of EGFR-TKI-advanced EGFR-mutant NSCLCs experienced grade 3/4 irAEs, and 30-40% were discontinued to osimertinib and ICI therapy combined due to irAEs [149].Concurrent osimertinib and ICI therapy were associated with an increased incidence of irAEs, leading to the early termination of a Phase III CAURAL recruitment [150].Interstitial lung disease (ILD) was a severe adverse response to EGFR TKIs [151].Osimertinib treatment that immediately followed prior ICI therapy also caused a high incidence of ILD [152,153].A meta-analysis of eight studies further concluded that a higher chance of irAEs was observed in EGFR-TKI plus ICI therapy in EGFR-TKI-advanced EGFR-mutant NSCLCs [154].
Other than toxicity concerns, worse clinical outcomes were seen in EGFR-mutant NSCLCs treated with EGFR TKI and ICI combined than those treated with EGFR TKI alone.The Phase Ib TATTON clinical trial showed that osimertinib and ICI combination therapy only achieved 43% ORR in the EGFR-TKI-advanced EGFR-mutant NSCLCs [149].In the Phase III CAURAL clinical trial, decreased ORR was also seen in the osimertinib plus ICI group vs. osimertinib alone [150].Similarly, the ORR in gefitinib plus ICI therapy (14.3%) was much worse than the 55% RR in gefitinib alone for EGFR-mutant NSCLC patients [155].In contrast to gefitinib plus ICI therapy, the untreated stage IIIB/IV EGFR-mutant NSCLCs tolerated erlotinib plus ICI therapy and better ORR (41.7%) in the KEYNOTE-021 [147].The 41.7% ORR was much lower than erlotinib alone (~70%) [156,157].

Hyper-Progressive Disease (HPD)
Hyper-progressive disease (HPD), characterized by unexpected fast tumor enlargement, both at rate and volume and early fatality of patients [158,159], was observed in 13.8% of NSCLC patients during treatment with ICI therapy in a retrospective study [160].By contrast, around 20% of EGFR-mutant NSCLCs showed risk of HPD after ICI therapy [159].

Conclusions
So far, ICI-based immunotherapy is ineffective for treatment-naïve EGFR-mutant NSCLC patients.Whether EGFR-TKI-advanced EGFR-mutant NSCLC patients may benefit from the ICI-based immunotherapy is worth further investigation, considering there is no approved effective treatment for this population.IrAEs will be an unmet roadblock during the evaluation.IrAEs may indicate the immune system is awakened.However, it is hard to predict irAEs, and some strong irAEs, if not controlled, may be deadly.A better understanding of the relationship of irAEs and efficacy may be helpful for future clinical monitoring during immunotherapy application.Other than irAEs, many other questions are still unsolved.Who may benefit from the immunotherapy: PD-L1 TPS > 50%, smoking history, high tumor mutation burden, high CD8 + T cells, and/or specific subtypes of EGFR mutations?Immunotherapy is not limited to anti-PD-1/PD-L1 monoclonal antibodies and could go beyond them.Additionally, the combination of ICI-based immunotherapy and several other treatment modalities is under active investigation.

Funding:
The review was supported by Emory University Lung Cancer SPORE, P50CA217691, funded by the National Cancer Institute (NCI) of the National Institute of Health.The content is solely the responsibility of the author and does not necessarily represent the official views of the NIH.Institutional Review Board Statement:The content is solely the responsibility of the author and does not necessarily represent the official views of the NIH.Informed Consent Statement: All discussed human-related studies were all from published studies, and related published studies were referenced behind each study.Data Availability Statement: Not applicable.Conflicts of Interest: Dan Yan is one of the inventors on a patent (number US10709708B2 (to Emory University)) describing the use of MRX-2843 in combination with osimertinib.